Project Summary Bacteriophages have evolved for many years with their bacterial hosts, and recent evidence shows that this battle has been ongoing in the human microbiome. There are 38 trillion bacteria that live in and on the human body, and they are accompanied by at least 10 times as many viruses. Many of those viruses are bacteriophages (phages). We now know that much of the unique diversity found in the human microbiome is imparted by those phages, which help to generate bacterial diversity as their hosts evolve to subvert phage attacks. The fact that the human body is inhabited by so many phages belies how successfully phages have evolved to identify and kill their hosts in humans. Phages have recently gained renewed interest with the rise of antimicrobial resistance (AMR) in bacteria. There are several different bacteria such as Carbapenem Resistant Enterobacteriaceae and Vancomycin Resistant Enterococcus that develop or acquire antibiotic resistance, becoming resistant to numerous commonly prescribed antibiotics. Because antibiotics can significantly alter the microbiome of humans (potentially resulting in significant long term health consequence), and the significant recent rises in AMR, the focus in developing therapeutics against bacterial pathogens has shifted to microbiome and AMR sparing alternatives such as phages. Understanding how we may uses phages to eradicate pathogens such as Enterococcus that live in our microbiomes, yet sometimes can become highly antibiotic resistant pathogens, has become of critical importance. We have assembled a multidisciplinary group of researchers with expertise in host-phage interactions (Dr. Whiteson), and clinical microbiology and the human microbiome (Dr. Pride) to isolate and identify phages effective against this understudied pathogen. We have preliminary data indicating that Enterococcus evolves in predictable ways to avoid phages, which we plan to take advantage of to identify characteristics of phages, phage cocktails, and phage-antibiotic combinations that work cooperatively to eradicate this pathogen. In this project, we will address three main questions. 1. Can we identify lytic Enterococcus phages from the human microbiome and do phages isolated from humans have broad range across Enterococcus species? 2. Can we identify the different mechanisms of resistance that bacterial hosts evolve against Enterococcus phages, and can these mechanisms inform cocktail design? 3. Will the use of antibiotics that have similar targets as some Enterococcus phages work synergistically to produce more effective therapeutic combinations? By addressing these three critical questions, we can significantly advance our knowledge of host-phage interactions, develop synergistic antibiotic-phage and phage-phage cocktails, and identify phage combinations that significantly reduce the emergence of bacteria resistant to phages and antibiotics. Our approach has the potential to help combat the bacteria responsible for debilitating and costly infections.